PROJECT SUMMARY/ ABSTRACT Normal physiology relies on the precise coordination of intrinsic cues, in the form of intracellular signal transduction pathways, with extrinsic cues like nutrient availability to balance cell growth and cell death. Transition metals such as copper (Cu) are tightly regulated micronutrients that function as structural or catalytic cofactors for proteins that are critical for normal physiology and development. Aberrant Cu excretion and absorption are manifested in the extremely rare genetic diseases Wilson and Menkes, respectively. The importance of intact Cu homeostatic mechanisms to cell growth control is underscored by the stunted growth and failure to thrive associated with Cu deficiency in Menkes disease patients and the prevalence of cancer in patients with hereditary Cu overload in Wilson disease. Further, Cu is neither created nor destroyed, and therefore low Cu dietary intake may be a contributing factor in impaired wound healing, cardiovascular disease, and non-alcoholic fatty liver disease. However, the dysregulation of a handful of currently identified Cu- dependent enzymes does not fully explain the diverse growth phenotypes associated with alterations in Cu metabolism. Thus, the direct cellular pathways that respond to and or/sense Cu abundance and are integrated to influence cellular proliferation remain undefined. Recent work by our group uncovered an unexpected link between the cellular acquisition of Cu and a mitogenic signaling cascade. In response to proliferative signals, Cu contributes to the amplitude of canonical MAPK signaling through a direct interaction between Cu and the kinases MEK1 and MEK2. This is the first example of Cu directly regulating the activity of a mammalian kinase and has exposed a new signaling paradigm that directly connects Cu to signaling pathway components. Based on our expertise, our group seeks to define the Cu-responsive and -sensing kinase signal transduction pathways to determine the mechanisms by which Cu contributes to pro-proliferative cellular processes that are essential to normal proliferation and are sustained during tumorigenesis. To accomplish our goals, we will utilize a multidisciplinary approach, which includes in vivo mouse models, biochemistry, biophysics, molecular biology, functional genomics, and pharmacologic interventions. Specifically, we will: i) elucidate the molecular mechanisms and cellular contexts that underlie Cu integration into the MAPK pathway, ii) systematically map Cu utilization by pro-proliferative kinase signal transduction pathways, and iii) leverage our experimental approaches and findings to other transition metals and kinase signaling networks in normal homeostasis and cancer. Completion of these studies has the potential to establish Cu availability as an integral component of intracellular communication and elucidate the molecular mechanism underlying this unique connection. Further, identifying novel Cu-dependent kinases can be therapeutically exploited to perturb Cu availability for essential signaling pathways in cancer and other diseases settings.